This invention relates to the drying of biological specimens prior to microscopic examination, and in particular to biological specimen preparation for the scanning electron microscope.
Scanning electron microscopes utilize electrons to visualize and object of study rather than light waves as in standard optical microscopy. They differ from the transmission electron microscope in that instead of electrons passing through the specimen and then striking a phosphor screen to produce an image, the electrons strike the specimen under investigation and cause a secondary electron emission. This secondary electron emission forms the basis for a high resolution video display of the specimen. To aid in the development of the secondary electron emission specimens usually have a metal or carbon coating vapor deposited on their surfaces prior to being studied in the scanning electron microscope.
The striking "three dimensional" visual effects observed make this form of microscopy of special interest to the lift scientist. Great insight into in vivo structure can be obtained. In order to take full advantage of scanning electron microscopy high quality methods for preserving specimen morphology have been devised, such as the technique called "Critical Point Drying". In critical point drying specimens for study are first subjected to a graded series of drying solvents such as alcohol/water or acetone/water, finally progressing to 100% alcohol or acetone. The alcohol or acetone is then usually replaced with amyl acetate, and the amyl acetate saturated specimens are placed within the critical point dryer.
At this point a transitional fluid such as liquid carbon dioxide, nitrous oxide, or trifluorochloromethane is slowly fed into the critical point dryer to displace the amyl acetate. The pressure sealed dryer is then heated to bring the apparatus to the critical temperature and pressure. The now gaseous transitional fluid surrounds the specimen and may be bled off without surface tension distortion of the specimen's surface. The dried specimen is removed from the critical point dryer, vacuum coated with a metal or carbon, and may now be studied in the scanning electron microscope.
I have discovered that specimen preservation results directly comparable or even superior to critical point drying may be obtained in a greatly simplified manner. In my method alcohol or acetone dried specimens are placed in a fluorocarbon transitional fluid, the fluorocarbon liquid is frozen, and then vacuum sublimated, leaving a dry specimen of equivalent or superior quality morphological preservation to that obtained with critical point drying, but without the necessity for costly critical point drying instrumentation and the time consuming procedures required.
Accordingly it is an object of this invention to provide a simple method for drying specimens to be examined in the scanning electron microscope.
Another object is to reduce equipment requirements for scanning electron microscope sample preparation.
Another object is to provide a rapid method for preparing quantities of specimens for scanning electron microscope observation.
A further object is to provide efficient, selective specimen lipid removal in a relatively inert environment.